This application claims the benefit of the following provisional applications: U.S. Ser. No. 60/215,986, filed Jul. 5, 2000 and U.S. Ser. No. 60/277,012, filed Mar. 19, 2001 under 35 USC 119(e)(i).
The present invention provides pyrroloquinolones that are useful as antiviral agents, more specifically, provides compounds of formula (I) described herein below against herpesviruses.
The herpesviruses comprise a large family of double stranded DNA viruses. They are also a source of the most common viral illnesses in man. Eight of the herpesviruses, herpes simplex virus types 1 and 2 (HSV-1 and HSV-2), varicella zoster virus (VZV), human cytomegalovirus (HCMV), Epstein-Barr virus (EBV), and human herpesviruses 6, 7, and 8 (HHV-6, HHV-7, and HHV-8), have been shown to infect humans. HSV-1 and HSV-2 cause herpetic lesions on the lips and genitals, respectively.
They also occasionally cause infections of the eye and encephalitis. HCMV causes birth defects in infants and a variety of diseases in immunocompromised patients such as retinitis, pneumonia, and gastrointestinal disease. VZV is the causitive agent of chicken pox and shingles. EBV causes infectious mononucleosis. It can also cause lymphomas in immunocompromised patients and has been associated with Burkitt""s lymphoma, nasopharyngeal carcinoma, and Hodgkins disease. HHV-6 is the causative agent of roseola and may be associated with multiple sclerosis and chronic fatigue syndrome. HHV-7 disease association is unclear, but it may be involved in some cases of roseola. HHV-8 has been associated with Karposi""s sarcoma, body cavity based lymphomas, and multiple myeloma.
Due to the unique position of the substitutent on the N-phenylmethyl of formula I described herein below, compounds of the present invention demonstrate unexpected activity against the above reference herpesviral infections, particularly, human cytomegaloviral infection.
PCT publication WO 97/31000 discloses pyrroloquinolone carboxamides useful as antiviral agents.
PCT publication WO 97/30999 discloses pyrroloquinolones with bicyclic carboxamides useful as antiviral agents.
U.S. Pat. No. 3,917,609 discloses pyrroloquinoline carboxylic acid derivatives useful as antiviral agents.
U.S. Pat. No. 4,547,511 discloses heterocyclic carboxamides which increase the activity of the immune system.
U.S. Pat. No. 4,317,820 discloses O-lactam series compound useful as antibacterial agents.
U.S. Pat. No. 5,792,774 discloses quinolones and their therapeutic use.
PCT publication WO 91/05783 discloses heterocyclic compounds that are 5-HT3 antagonists.
PCT publication WO 92/18483 discloses quinoline derivatives.
Abstract of Japanese patent J55145-612 discloses antimicrobial agent contains a novel xcex2-lactam ring.
Abstract of Japanese patent J55153-792 discloses cephalosporanic derivatives.
GB 2236751 A discloses 4-oxo-quinolines which are 5-HT3 antagonists.
Blurton, et. al Heterocycles, 1997, 45, 2395 discloses the preparation of pyrroloquinolones.
The present invention provides a compound of formula I, 
or a pharmaceutically acceptable salt, racemate, solvate, tautomer, optical isomer or prodrug derivative thereof wherein:
R1 is F, Cl, Br, CN or NO2;
R2 and R3 are independently H, halo, OR11, C(xe2x95x90O)R7, C(xe2x95x90O)OR11, C3-8cycloalkyl, C1-7alkyl which may be partially unsaturated and optionally substituted by one or more halo, C3-8cycloalkyl, R2, OR14, SR11, SR14, NR8R9, NR11C(O)R7, (Cxe2x95x90O)C1-7 alkyl, or SOmR10;
R4 and R5 are independently
(a) H,
(b) halo,
(c) aryl,
(d) S(O)mR7,
(e) (Cxe2x95x90O)R7,
(f) (Cxe2x95x90O)OR10,
(g) CN,
(h) het, wherein said het is bound via a carbon atom,
(i) OR11,
(j) Ohet,
(k) NR8R9 
(1) SR11,
(m) Shet,
(n) NHCOR13,
(O)NHSO2R13,
(p) C3-8cycloalkyl, or
(q) C1-7alkyl which may be partially unsaturated and optionally substituted by one or more R12, OR14, SR11, SR14, NR8R9, halo, C3-8cycloalkyl, (Cxe2x95x90O)C1-7alkyl, or SOmR10;
R is H, halo, C3-8cycloalkyl, or C1-4alkyl optionally substituted by 1-3 halo;
R7 is
(a) C1-7alkyl,
(b) C3-8cycloalkyl,
(c) NR8R9,
(d) aryl, or
(e) het, wherein said het is bonded via a carbon atom;
R8 and R9 are independently
(a) H,
(b) aryl,
(c) C3-8cycloalkyl,
(d) C1-7alkyl which may be partially unsaturated and is optionally substituted by one or more NR11R11, R12, SOmR10, CONR11R11, OH, aryl, het, C3-8cycloalkyl, or halo, or
(e) R8 and R9 together with the nitrogen to which they are attached for a het;
R10 is
(a) aryl,
(b) het,
(c) C3-8cycloalkyl, or
(d) C1-7alkyl which may be partially unsaturated and is optionally substituted by one or more NR11R11, R12, SH, CONR11R11, C3-8cycloalkyl, or halo;
R11 is
(a) H,
(b) C3-8cycloalkyl,
(c) C1-7alkyl optionally substituted by OH, or
(d) aryl;
R12 is
(a) OR11,
(b) Ohet,
(c) Oaryl,
(d) CO2R11,
(e) het,
(f) aryl, or
(g) CN;
R13 is
(a) H,
(b) het,
(c) aryl,
(d) C3-8cycloalkyl, or
(e) C1-7alkyl optionally substituted by NR11R11 or R12;
R14 is
(a) (Pxe2x95x90O)(OR15)2,
(b) CO(CH2)nCON(CH3)xe2x80x94(CH2)nSO3xe2x88x92M+,
(c) an amino acid,
(d) C(xe2x95x90O)aryl,
(e) C(xe2x95x90O)C1-7alkyl optionally substituted by NR11R11, aryl, het, CO2H, or O(CH2)nCO2R15;
R15 is
(a) H, or
(b) C1-7alkyl;
aryl is a phenyl radical or an ortho-fused bicyclic carbocyclic radical wherein at least one ring is aromatic; at each occurrence, aryl may be additionally substituted with one or more halo, CN, CO2R11, SR11, OR11, NR11R11, C1-4alkyl, CF3, or C3-8cycloalkyl;
het is a fourxe2x80x94(4), fivexe2x80x94(5), sixxe2x80x94(6), or sevenxe2x80x94(7) membered saturated or unsaturated heterocyclic ring having 1, 2, or 3 heteroatoms selected from the group consisting of O, SOm, and NX; wherein X is H, C1-4alkyl or absence, wherein het is optionally fused to a benzene ring, or any bicyclic heterocycle group; at each occurrence, het may be additionally substituted with one or more halo, CN, CO2R11, COR13, SR11, OR11, NR11R11, C1-4alkyl, CF3, C3-8cycloalkyl, oxo or oxime;
at each occurrence, C3-8cycloalkyl may be partially unsaturated and optionally substituted by one or more R12, SR11, NR11R11, CONR11R11, or halo;
m is 0, 1, or 2; and
at each occurrence n is independently 1, 2, 3, 4, 5 or 6;
M is sodium, potassium, or lithium.
The present invention further provides a pharmaceutical composition comprising a compound of formula I, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier (the composition preferably comprises an effective antiviral amount of the compound or salt).
The present invention further provides a method of treating or preventing a herpesviral infection, comprising administering to a mammal in need of such treatment, a compound of formula (I) or a pharmaceutically acceptable salt thereof.
The present invention further provides a method of treating or preventing a herpesviral infection comprising administering orally, parenterally, topically, rectally, nasally, sublingually or transdermally an effective amount of a compound of claim 1.
The present invention further provides a compound of formula (I) or a pharmaceutically acceptable salt thereof for use in medical treatment.
The present invention further provides the use of a compound of formula (I) or a pharmaceutically acceptable salt thereof to prepare a medicament for treating or preventing a herpesviral infection in a mammal.
The present invention further provides a method for inhibiting a viral DNA polymerase, comprising contacting (in vitro or in vivo) the polymerase with an effective inhibitory amount of a compound of formula I, or a pharmaceutically acceptable salt thereof.
The invention also provides novel intermediates and processes disclosed herein that are useful for preparing compounds of formula I.
The following definitions are used, unless otherwise described. Halo denotes fluoro, chloro, bromo, or iodo. Alkyl, alkoxy, etc. denote both straight and branched groups; but reference to an individual radical such as xe2x80x9cpropylxe2x80x9d embraces only the straight chain radical, a branched chain isomer such as xe2x80x9cisopropylxe2x80x9d being specifically referred to. When alkyl can be partially unsaturated, the alkyl chain may comprise one or more (e.g. 1, 2, 3, or 4) double or triple bonds in the chain.
The carbon atom content of various hydrocarbon-containing moieties is indicated by a prefix designating the minimum and maximum number of carbon atoms in the moiety, i.e., the prefix Ci-j indicates a moiety of the integer xe2x80x9cixe2x80x9d to the integer xe2x80x9cjxe2x80x9d carbon atoms, inclusive. Thus, for example, (C1-3)alkyl refers to alkyl of one to three carbon atoms, inclusive, or methyl, ethyl, propyl and isopropyl, straight and branched forms thereof.
Aryl is a phenyl radical or an ortho-fused bicyclic carbocyclic radical wherein at least one ring is aromatic. Aryl is optionally substituted with one or more substituents selected from the group consisting of halo, CN, CO2R11, SR11, OR11, NR11R11, C1-4alkyl, CF3, or C3-8cycloalkyl. Preferably, aryl is phenyl, or naphthalene.
Het is a fourxe2x80x94(4), fivexe2x80x94(5), sixxe2x80x94(6), or sevenxe2x80x94(7) membered saturated or unsaturated heterocyclic ring having 1, 2, or 3 heteroatoms selected from the group consisting of oxygen (O), sulfur (Sm, m is 0, 1, or 2), and nitrogen (NX wherein X is H, C1-4alkyl or absence), which is optionally fused to a benzene ring, or any bicyclic heterocycle group. Het is optionally substituted with one or more substituents selected from the group consisting of halo, CN, CO2R11, COR13, SR11, OR11, NR11R11, C1-4alkyl, CF3, C3-8cycloalkyl, oxo or oxime.
The term xe2x80x9chetxe2x80x9d includes piperidinyl, morpholinyl, thiomorpholinyl, 1,1-dioxidothiomorpholinyl, piperazinyl, Nxe2x80x94C1-4alky substituted piperazinyl such as 4-methyl piperazinyl, pyrrolidinyl, pyridyl, imidazolyl, Nxe2x80x94C1-4alky substituted imidazole such as 1-methyl-1H-imidazole, thiophene, furan, pyrazoline, pyrimidine, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidinyl, 4-pyrimidinyl, 5-pyrimidinyl, 3-pyridazinyl, 4-pyridazinyl, 3-pyrazinyl, 2-quinolyl, 3-quinolyl, 1-isoquinolyl, 3-isoquinolyl, 4-isoquinolyl, 2-quinazolinyl, 4-quinazolinyl, 2-quinoxalinyl, 1-phthalazinyl, 4-oxo-2-imidazolyl, 2-imidazolyl, 4-imidazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 3-pyrazolyl, 4-pyrazolyl, 5-pyrazolyl, 2-oxazolyl, 4-oxazolyl, 4-oxo-2-oxazolyl, 5-oxazolyl, 4,5,-dihydrooxazole, 1,2,3-oxathiole, 1,2,3-oxadiazole, 1,2,4-oxadiazole,
1,2,5-oxadiazole, 1,3,4-oxadiazole, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 3-isothiazole, 4-isothiazole, 5-isothiazole, 2-indolyl, 3-indolyl, 3-indazolyl, 2-benzoxazolyl, 2-benzothiazolyl, 2-benzimidazolyl, 2-benzofuranyl, 3-benzofuranyl, benzoisothiazole, benzisoxazole, 2-furanyl, 3-furanyl, 2-thienyl, 3-thienyl, 2-pyrrolyl, 3-pyrrolyl, 3-isopyrrolyl, 4-isopyrrolyl, 5-isopyrrolyl, 1,2,3,-oxathiazole-1-oxide, 1,2,4-oxadiazol-3-yl, 1,2,4-oxadiazol-5-yl, 5-oxo-1,2,4-oxadiazol-3-yl, 1,2,4-thiadiazol-3-yl, 1,2,4-thiadiazol-5-yl, 3-oxo-1,2,4-thiadiazol-5-yl, 1,3,4-thiadiazol-5-yl, 2-oxo-1,3,4-thiadiazol-5-yl, 1,2,4-triazol-3-yl, 1,2,4-triazol-5-yl, 1,2,3,4-tetrazol-5-yl, 5-oxazolyl, 1-pyrrolyl, 1-pyrazolyl, 1,2,3-triazol-1-yl, 1,2,4-triazol-1-yl, 1-tetrazolyl, 1-indolyl, 1-indazolyl, 2-isoindolyl, 7-oxo-2-isoindolyl, 1-purinyl, 3-isothiazolyl, 4-isothiazolyl and 5-isothiazolyl, 1,3,4,-oxadiazole, 4-oxo-2-thiazolinyl, or 5-methyl-1,3,4-thiadiazol-2-yl, thiazoledione, 1,2,3,4-thiatriazole, 1,2,4-dithiazolone. Each of these moieties may be substituted as appropriate.
The term xe2x80x9chetxe2x80x9d also includes azetidyl, tetrahydrofuranyl, dioxolanyl, imidazolidinyl, oxathiolanyl, oxazolidinyl.
The term xe2x80x9chetxe2x80x9d also includes pyran, thiopyran, tetrahydropyran or tetrahydrothiopyran.
xe2x80x9cAmino acid,xe2x80x9d includes a residue of natural amino acid (e.g. Ala, Arg, Asn, Asp, Cys, Glu, Gln, Gly, His, Hyl, Hyp, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val) in D or L form, as well as unnatural amino acids (e.g. phosphoserine, phosphothreonine, phosphotyrosine, hydroxyproline, gamma-carboxyglutamate; hippuric acid, octahydroindole-2-carboxylic acid, statine, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, penicillamine, ornithine, citruline, -methyl-alanine, para-benzoylphenylalanine, phenylglycine, propargylglycine, sarcosine, and tert-butylglycine). An amino acid can conveniently be linked to the remainder of a compound of formula I through the carboxy terminus, the amino terminus, or through any other convenient point of attachment, such as, for example, through the sulfur of cysteine. In particular, an amino acid can conveniently be linked to the remainder of a compound of formula I through the carboxy terminus.
Mammal denotes human and animals. Animals specifically refers to food animal or companion animal.
It will be appreciated by those skilled in the art that compounds of the invention may have one or more chiral centers and be isolated in optically active and racemic forms. Some compounds may exhibit polymorphism. It is to be understood that the present invention encompasses any racemic, optically-active, polymorphic, tautomeric, or stereoisomeric form, or mixture thereof, of a compound of the invention, which possesses the useful properties described herein, it being well known in the art how to prepare optically active forms (for example, by resolution of the racemic form by recrystallization techniques, by synthesis from optically-active starting materials, by chiral synthesis, or by chromatographic separation using a chiral stationary phase) and how to determine antiviral activity using the standard tests described herein, or using other similar tests which are well known in the art.
The compounds of the present invention are generally named according to the IUPAC or CAS nomenclature system. Abbreviations which are well known to one of ordinary skill in the art may be used (e.g. xe2x80x9cPhxe2x80x9d for phenyl, xe2x80x9cMexe2x80x9d for methyl, xe2x80x9cEtxe2x80x9d for ethyl, xe2x80x9chxe2x80x9d for hour or hours and xe2x80x9crtxe2x80x9d for room temperature).
Specific and preferred values listed below for radicals, substituents, and ranges, are for illustration only; they do not exclude other defined values or other values within defined ranges for the radicals and substituents.
Specifically, the term xe2x80x9cC1-8alkyl,xe2x80x9d or xe2x80x9cC1-4alkylxe2x80x9d refers to an alkyl group having one to eight or one to four carbon atoms such as, for example, methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, and their isomeric forms thereof.
Specifically, a 5- or 6-membered heterocyclic ring includes piperidinyl, morpholinyl, thiomorpholinyl, piperazinyl, Nxe2x80x94C1-4alkyl substituted piperazinyl such as 4-methyl piperazinyl, or pyrrolidinyl.
Specifically, a 5- or 6-membered heterocyclic ring includes pyridyl, imidazolyl, Nxe2x80x94C1-4alkyl substituted imidazole such as 1-methyl-1H-imidazole.
Specifically, R1 is Cl.
Specifically, R4 and R6 are hydrogen.
Specifically, R5 is C1-7alkyl which may be partially unsaturated and optionally substituted by OH.
Specifically, R5 is C1-7alkyl substituted by het.
Specifically, R5 is C1-7alkyl substituted by piperidinyl, morpholinyl, thiomorpholinyl, piperazinyl, pyrrolidinyl, pyridyl, imidazolyl, or tetrahydro-2H-pyran.
Specifically, R5 is C1-7alkyl substituted by morpholinyl.
Specifically, R5 is C1-7alkyl substituted by tetrahydro-2H-pyran.
Specifically, R5 is C1-7alkyl substituted by NR8R9.
Specifically, R8 and R9 are independently H, or C1-6alkyl optionally substituted by one to three OH, SH, halo, phenyl, or het.
Specifically, R8 and R9 are independently H, or C1-6alkyl optionally substituted by one to two OH, or phenyl.
Specifically, R2 and R3 are independently hydrogen, or C1-7alkyl which may be partially unsaturated and optionally substituted by OH.
Specifically, R3 is hydrogen.
Specifically, R3 is halo.
Specifically, R2 is hydroxymethyl.
Specifically, R2 is hydroxyethyl.
Specifically, R2 is C1-7alkyl substituted by het.
Specifically, R2 is C1-7alkyl substituted by piperidinyl, morpholinyl, thiomorpholinyl, piperazinyl, pyrrolidinyl, pyridyl, imidazolyl, or tetrahydro-2H-pyran.
Specifically, R2 is C1-7alkyl substituted by 2-ethylpiperidinyl, 1,1-dioxido-4-thiomorpholinyl, 4-methylpiperazinyl.
Specifically, R2 is C1-7alkyl substituted by NR8R9.
Specifically, R8 and R9 are independently H, or C1-6alkyl optionally substituted by one to three OH, SH, CONR11R11, phenyl, or het, wherein each R11 is independently H, or C1-6alkyl.
Specifically, R2 is C1-7alkyl substituted by OR11 or SR11.
Specifically, R11 is C1-4 alkyl or phenyl.
Specifically, a compound of the present invention is where R1 is Cl; R2 is hydrogen, or C1-7alkyl which may be partially unsaturated and optionally substituted by OR11, het, or NR8R9; R3 is hydrogen or halo; R4 and R6 are hydrogen; and R5 is C1-7alkyl which may be partially unsaturated and optionally substituted by OH, het, or NR8R9.
Examples of the compounds of the present invention are:
(a) N (4-chlorobenzyl)-2-(hydroxymethyl)-6-oxo-6H-pyrrolo[3,2,1-ij]quinoline-5-carboxamide;
(b) N-(4-chlorobenzyl)-2-(hydroxymethyl)-6-oxo-8-(tetrahydro-2H-pyran-4-ylmethyl)-6H-pyrrolo[3,2,1-ij]quinoline-5-carboxamide;
(c) N-(4-chlorobenzyl)-2-(hydroxymethyl)-8-(4-morpholinylmethyl)-6-oxo-6H-pyrrolo[3,2,1-ij]quinoline-5-carboxamide;
(d) N-(4-chlorobenzyl)-2-(2-hydroxyethyl)-8-(4-morpholinylmethyl)-6-oxo-6H-pyrrolo[3,2,1-ij]quinoline-5-carboxamide;
(e) N-(4-chlorobenzyl)-2-(2-morpholin-4-ylethyl)-8-(4-morpholinylmethyl)-6-oxo-6H-pyrrolo[3,2,1-ij]quinoline-5-carboxamide;
(f) N-(4-chlorobenzyl)-2-[2-(diethylamino)ethyl]-8-(4-morpholinylmethyl)-6-oxo-6H-pyrrolo[3,2,1-ij]quinoline-5-carboxamide;
(g) N-(4-chlorobenzyl)-2-[2-(4-methylpiperazin-1-yl)ethyl]-8-(morpholin-4-ylmethyl)-6-oxo-6H-pyrrolo[3,2,1-ij]quinoline-5-carboxamide;
(h) N-(4-chlorobenzyl)-2-[2-(2-ethylpiperidin-1-yl)ethyl]-8-(morpholin-4-ylmethyl)-6-oxo-6H-pyrrolo[3,2,1-ij]quinoline-5-carboxamide;
(i) N-(4-chlorobenzyl)-2-[3-(4-methylpiperazin-1-yl)propyl]-8-(morpholin-4-ylmethyl)-6-oxo-6H-pyrrolo[3,2,1-ij]quinoline-5-carboxamide;
(j) N-(4-chlorobenzyl)-8-(morpholin-4-ylmethyl)-6-oxo-2-(2-piperidin-1-ylethyl)-6H-pyrrolo[3,2,1-ij]quinoline-5-carboxamide;
(k) N-(4-chlorobenzyl)-8-(morpholin-4-ylmethyl)-2-(3-morpholin-4-ylpropyl)-6-oxo-6H-pyrrolo[3,2,1-ij]quinoline-5-carboxamide;
(1) N-(4-chlorobenzyl)-2-[(1,1-dioxido-4-thiomorpholinyl)methyl]-8-(4-morpholinylmethyl)-6-oxo-6H-pyrrolo[3,2,1-ij]quinoline-5-carboxamide;
(m) N-(4-chlorobenzyl)-2-[(1,1-dioxido-4-thiomorpholinyl)methyl]-1-iodo-8-(4-morpholinylmethyl)-6-oxo-6H-pyrrolo[3,2,1-ij]quinoline-5-carboxamide;
(n) N-(4-chlorobenzyl)-2-(3-hydroxypropyl)-8-(4-morpholinylmethyl)-6-oxo-6H-pyrrolo[3,2,1-ij]quinoline-5-carboxamide;
(O) 2-{[(aminocarbonyl)amino]methyl}-N-(4-chlorobenzyl)-8-(4-morpholinylmethyl)-6-oxo-6H-pyrrolo[3,2,1-ij]quinoline-5-carboxamide;
(p) N-(4-chlorobenzyl)-2-[(1R)-1-hydroxyethyl]-8-(4-morpholinylmethyl)-6-oxo-6H-pyrrolo[3,2,1-ij]quinoline-5-carboxamide;
(q) N-(4-chlorobenzyl)-2-(methoxymethyl)-8-(4-morpholinylmethyl)-6-oxo-6H-pyrrolo[3,2,1-ij]quinoline-5-carboxamide;
(r) N-(4-chlorobenzyl)-2-[(ethylsulfanyl)methyl]-8-(4-morpholinylmethyl)-6-oxo-6H-pyrrolo[3,2,1-ij]quinoline-5-carboxamide;
(s) N-(4-chlorobenzyl)-8-(4-morpholinylmethyl)-6-oxo-2-[(phenylsulfanyl)methyl]-6H-pyrrolo[3,2,1-ij]quinoline-5-carboxamide;
(t) N-(4-chlorobenzyl)-2-[(methylamino)methyl]-8-(4-morpholinylmethyl)-6-oxo-6H-pyrrolo[3,2,1-ij]quinoline-5-carboxamide;
(u) N-(4-chlorobenzyl)-2-[(dimethylamino)methyl]-8-(4-morpholinylmethyl)-6-oxo-6H-pyrrolo[3,2,1-ij]quinoline-5-carboxamide;
(v) N-(4-chlorobenzyl)-2-(2-hydroxyethyl)-6-oxo-6H-pyrrolo[3,2,1-ij]quinoline-5-carboxamide; or
(w) N-(4-chlorobenzyl)-2-(2-hydroxyethyl)-6-oxo-8-(tetrahydro-2H-pyran-4-ylmethyl)-6H-pyrrolo[3,2,1-ij]quinoline-5-carboxamide.
Other examples of the present invention are
(a) N-(4-chlorobenzyl)-2-[2-hydroxy-1-(hydroxymethyl)ethyl]-8-(4-morpholinylmethyl)-6-oxo-6H-pyrrolo[3,2,1-ij]quinoline-5-carboxamide;
(b) N-(4-chlorobenzyl)-2-[1,2-dihydroxy-1-(hydroxymethyl)ethyl]-8-(4-morpholinylmethyl)-6-oxo-6H-pyrrolo[3,2,1-ij]quinoline-5-carboxamide;
(c) N-(4-chlorobenzyl)-2-[(ethylsulfonyl)methyl]-8-(4-morpholinylmethyl)-6-oxo-6H-pyrrolo[3,2,1-ij]quinoline-5-carboxamide;
(d) N-(4-chlorobenzyl)-2-[(ethylsulfinyl)methyl]-8-(4-morpholinylmethyl)-6-oxo-6H-pyrrolo[3,2,1-ij]quinoline-5-carboxamide;
(e) 2-{[bis(2-hydroxyethyl)amino]methyl}-N-(4-chlorobenzyl)-8-(4-morpholinylmethyl)-6-oxo-6H-pyrrolo[3,2,1-ij]quinoline-5-carboxamide;
(f) N-(4-chlorobenzyl)-2-[(2-hydroxyethoxy)methyl]-8-(4-morpholinylmethyl)-6-oxo-6H-pyrrolo[3,2,1-ij]quinoline-5-carboxamide;
(g) N-(4-chlorobenzyl)-2-(1,2-dihydroxyethyl)-8-(4-morpholinylmethyl)-6-oxo-6H-pyrrolo[3,2,1-ij]quinoline-5-carboxamide;
(h) N-(4-chlorobenzyl)-8-(4-morpholinylmethyl)-6-oxo-2-(1,2,3-trihydroxypropyl)6H-pyrrolo[3,2,1-ij]quinoline-5-carboxamide;
(i) N-(4-chlorobenzyl)-2-[3-hydroxy-2-(hydroxymethyl)propyl]-8-(4-morpholinylmethyl)-6-oxo-6H-pyrrolo[3,2,1-ij]quinoline-5-carboxamide;
(j) N-(4-chlorobenzyl)-1-(hydroxymethyl)-8-(morpholin-4-ylmethyl)-6-oxo-6H-pyrrolo[3,2,1-ij]quinoline-5-carboxamide;
(k) N-(4-chlorobenzyl)-1-(2-hydroxyethyl)-8-(morpholin-4-ylmethyl)-6-oxo-6H-pyrrolo[3,2,1-ij]quinoline-5-carboxamide;
(l) N-(4-chlorobenzyl)-1-(3-hydroxypropyl)-8-(morpholin-4-ylmethyl)-6-oxo-6H-pyrrolo[3,2,1-ij]quinoline-5-carboxamide;
(m) N-(4-chlorobenzyl)-1-(2-morpholin-4-ylethyl)-8-(morpholin-4-ylmethyl)-6-oxo-6H-pyrrolo[3,2,1-ij]quinoline-5-carboxamide;
(n) N-(4-chlorobenzyl)-8-(morphol in-4-ylmethyl)-6-oxo-1-(2-thiomorpholin-4-ylethyl)-6H-pyrrolo[3,2,1-ij]quinoline-5-carboxamide;
(o) N-(4-chlorobenzyl)-1-[2-(1,1-dioxidothiomorpholin-4-yl)ethyl]-8-(morpholin-4-ylmethyl)-6-oxo-6H-pyrrolo[3,2,1-ij]quinoline-5-carboxamide;
(p) N-(4-chlorobenzyl)-1-[2-(4-methylpiperazin-1-yl)ethyl]-8-(morpholin-4-ylmethyl)-6-oxo-6H-pyrrolo[3,2,1-ij]quinoline-5-carboxamide;
(q) N-(4-chlorobenzyl)-8-(morpholin-4-ylmethyl)-6-oxo-1-(2-piperazin-1-ylethyl)-6H-pyrrolo[3,2,1-ij]quinoline-5-carboxamide;
(r) 1-[(acetylamino)methyl]-N-(4-chlorobenzyl)-8-(morpholin-4-ylmethyl)-6-oxo-6H-pyrrolo[3,2,1-ij]quinoline-5-carboxamide;
(s) N-(4-chlorobenzyl)-1-[(1S)-1-hydroxyethyl]-8-(morpholin-4-ylmethyl)-6-oxo-6H-pyrrolo[3,2,1-ij]quinoline-5-carboxamide;
(t) N-(4-chlorobenzyl)-1-(1H-imidazol-1-ylmethyl)-8-(morpholin-4-ylmethyl)-6-oxo-6H-pyrrolo[3,2,1-ij]quinoline-5-carboxamide;
(u) 1-(1H-1,2,3-benzotriazol-1-ylmethyl)-N-(4-chlorobenzyl)-8-(morpholin-4-ylmethyl)-6-oxo-6H-pyrrolo[3,2,1-ij]quinoline-5-carboxamide;
(v) N-(4-chlorobenzyl)-8-(morpholin-4-ylmethyl)-6-oxo-1-(pyridin-3-ylmethyl)-6H-pyrrolo[3,2,1-ij]quinoline-5-carboxamide;
(w) N-(4-chlorobenzyl)-8-(morpholin-4-ylmethyl)-6-oxo-1-{[({[3-(trifluoromethyl)phenyl]amino} carbonyl)amino]methyl}-6H-pyrrolo[3,2,1-ij]quinoline-5-carboxamide;
(x) N-(4-chlorobenzyl)-8-(4-morpholinylmethyl)-6-oxo-2-[2-(3-oxo-1-azetidinyl)ethyl]-6H-pyrrolo[3,2,1-ij]quinoline-5-carboxamide;
(y) N-(4-chlorobenzyl)-2-[2-(3-hydroxy-1-azetidinyl)ethyl]-8-(4-morpholinylmethyl)-6-oxo-6H-pyrrolo[3,2,1-ij]quinoline-5-carboxamide;
(z) N-(4-chlorobenzyl)-2-(2,3-dihydroxypropyl)-8-(4-morpholinylmethyl)-6-oxo-6H-pyrrolo[3,2,1-ij]quinoline-5-carboxamide;
(aa) N-(4-chlorobenzyl)-2-[(1S)-1-hydroxyethyl]-8-(4-morpholinylmethyl)-6-oxo-6H-pyrrolo[3,2,1-ij]quinoline-5-carboxamide;
(bb) N-(4-chlorobenzyl)-2-[2-(1H-imidazol-1-yl)ethyl]-8-(morpholin-4-ylmethyl)-6-oxo-6H-pyrrolo[3,2,1-ij]quinoline-5-carboxamide;
(cc) N-(4-chlorobenzyl)-2-[2-(1H-imidazol-2-yl)ethyl]-8-(morpholin-4-ylmethyl)-6-oxo-6H-pyrrolo[3,2,1-ij]quinoline-5-carboxamide;
(dd) N-(4-chlorobenzyl)-8-(morpholin-4-ylmethyl)-6-oxo-2-[2-(4H-1,2,4-triazol-3-yl)ethyl]-6H-pyrrolo[3,2,1-ij]quinoline-5-carboxamide;
(ee) N-(4-chlorobenzyl)-8-(morpholin-4-ylmethyl)-6-oxo-2-[2-(1H-tetraazol-5-yl)ethyl]-6H-pyrrolo[3,2,1-ij]quinoline-5-carboxamide;
(ff) N-(4-chlorobenzyl)-8-(morpholin-4-ylmethyl)-6-oxo-2-(2-piperazin-1-ylethyl)-6H-pyrrolo[3,2,1-ij]quinoline-5-carboxamide; or
(gg) tert-butyl 4-{2-[5-{[(4-chlorobenzyl)amino]carbonyl}-8-(morpholin-4-ylmethyl)-6-oxo-6H-pyrrolo[3,2,1-ij]quinolin-2-yl]ethyl} piperazine-1-carboxylate.
The following Charts A-J describe the preparation of the compounds of formula I of the present invention. All of the starting materials are prepared by procedures described in these charts, by procedures well known to one of ordinary skill in organic chemistry or can be obtained commercially. All of the final compounds of the present invention are prepared by procedures described in these charts or by procedures analogous thereto, which would be well known to one of ordinary skill in organic chemistry. In the Charts A to J, R is R4, R5, or R6 and i is one to three. Other variables used in the charts are as defined below or as in the claims.
As shown in Chart A, Sonogashira coupling of a 4-hydroxy-8-iodoquinoline with an acetylene employing PdCl2(PPh3)2 and copper iodide in diethylamine solvent and subsequent cyclization under the reaction conditions provides the desired pyrroloquinolones (Blurton, et. al Heterocycles, 1997, 45, 2395). 
The requisite 8-iodo-4-hydroxyquinolines can be prepared according to Chart B. 2-Iodoanilines (B.2) can either be purchased from commercial sources or prepared by iodination of an aniline using reagents such as ICl. Condensation of the resulting 2-iodoaniline with diethyl ethoxymethylenemalonate and subsequent cyclization either under thermal conditions or by heating in Eaton""s reagent provides the 4-hydroxy-8-iodoquinoline-3-carboxylate B.3. Amide formation can be accomplished by treatment with a neat amine (e.g. 4-chlorobenzylamine) at elevated temperatures to provide the 4-hydroxy-8-iodoquinoline-3-carboxamide B.4. 
Some of the substituted anilines can be prepared according to Chart C and Chart D. For carbon-substituted anilines, Wittig reaction between (4-nitrobenzyl)(triphenyl)-phosphonium bromide (C.2) and the desired aldehyde provides the nitrobenzylidine C.3. Hydrogenation over PtO2 reduces the nitro group and the olefin to provide the corresponding aniline C.4. Iodination and quinoline formation as described in Chart B then provides the desired 8-iodo-4-hydroxyquinoline C.7. 
Aminomethyl, hydroxymethyl, alkoxymethyl, etc. substituted analogs can be prepared as shown in Chart D. Iodination of 4-aminobenzoate using NIS provides the 2-iodoaniline D.2. Reduction of the ester with DIBAL-H provides the hydroxymethyliodoaniline D.3. Condensation of the aniline with neat diethyl ethoxymethylenemalonate at elevated temperatures provides the enamine D.4. Protection of the alcohol with acetic anhydride in acetic acid solvent gives D.5. Cyclization in refluxing diphenylether provides the 8-iodo-4-hydroxyquinoline-3-carboxylate D.6. Treatment with a neat amine (e.g. 4-chlorobenzylamine) at elevated temperatures results in amide formation and deprotection of the alcohol to give the hydroxymethyl quinoline D.7. At this point, the hydroxyl group can be alkylated or mesylated with methanesulfonyl chloride and displaced with a nucleophile (e.g. morpholine) to give the desired 8-iodoquinoline D.8. 
4-Nitrobenzylbromide (E.0) is treated with morpholine and potassium carbonate in acetone to give 4-(4-nitrobenzyl)morpholine (E.1) of Chart E. The nitro group is reduced with platinum on carbon and hydrogen gas to afford the aniline E.2. Iodination employing ICI or NIS provides the amine E.3 which is then treated with diethyl ethoxymethylenemalonate to give the enamine E.4. Cyclization with phosphorous pentoxide in methanesulfonic acid affords the quinoline ester E.5. Aminolysis with p-chlorobenzylamine at elevated temperatures gives the amide E.6. Coupling with an acetylene employing PdCl2(PPh3)2 and copper iodide and subsequent cyclization provides the desired pyrroloquinolones E.7. 
As shown in Chart F, palladium catalyzed heteroannulation of internal alkynes using appropriately substituted 2-iodoanilines F.0 provides the indoles F.1 (Larock et al. J. Am. Chem. Soc., 1991, 113, 6689). Reduction of the corresponding indoles provides the indolines F.2. Condensation of the indoline F.2 with diethyl ethoxymethylenemalonate affords enamine F.3. Cyclization using Eaton""s reagent provides ester F.4. Amide formation using neat amine gives amide F.5. Oxidation provides the corresponding indoles F.6. 
In Chart G alkylation of amide B.4 (preparation described in Chart B) with a suitably substituted propargyl bromide provides the N-propargyl-8-iodoquinoline-3-carboxamide G.1. Palladium catalyzed tandem cyclization-hydride ion capture (Grigg et al. Tetrahedron Lett., 1988, 34, 4325) provides the alkylidene indolines G.2. Isomerization of the exocylic G.2 double bond provides indoles G.3. 
A synthesis of compounds, where R2 and R3 of formula I are not hydrogen, is shown in Chart H (where X=Br or I). Compound H.1, synthesis given in Chart A, is iodinated or brominated to produce H.2. Application of the appropriate coupling protocol provides H.3. Nonexclusive examples of the appropriate couplings are the Suzuki, Stille and Negishi reactions. 
Chart I (where alkyl =C1-7 and Q=OC1-7 alkyl or C1-7 alkyl) describes the preparation of compounds 1.4. Alkoxycarbonylation of 1.0, previously described in Chart B, using a palladium catalyst contacted with CO in the presence of the appropriate alcohol provides ester 1.1. Nitrogen alkylation of 1.1 is accomplished by contacting with Cs2CO3 or K2CO3 or another suitable base in the presence of a bromo-, iodo- or chloromethyl ketone or 2-bromo, 2-iodo- or 2-chloroacetate to provide 1.2. 1.2 is converted to 1.3, or its keto variant, by Dieckmann cyclization in the presence of the appropriate base, for example KOt-Bu. Triflation of the hydroxy moiety in 1.3 followed by palladium, or another appropriate metal, catalyzed coupling yields 1.4. Alternatively, treatment of compounds occupying predominantly the keto form of 1.3 with a reactive nucleophile, for example a Grignard reagent, followed by acid catalyzed elimination also provides 1.4. 
For Chart J (where B=OC1-7 alkyl or C1-7 alkyl, and Z and L are subsets of R2 and R3), J.1 is prepared as described in Chart A with the appropriate propargyl alcohol. J.1 is converted into J.2 by first transient formation of an L and B substituted enol ether or ester enolate equivalent and subsequent rearrangement. A nonexclusive example of the [3.3]-sigmatropic rearrangement is the Claisen rearrangement. Selected J.2 compounds spontaneously convert to J.3 or are isomerized with acid catalysis. 
It will be apparent to those skilled in the art that the described synthetic procedures are merely representative in nature and alternative synthetic processes are known to one of ordinary skill in organic chemistry.
In cases where compounds are sufficiently basic or acidic to form stable nontoxic acid or base salts, administration of the compounds as salts may be appropriate. Examples of pharmaceutically acceptable salts are organic acid addition salts formed with acids which form a physiological acceptable anion, for example, tosylate, methanesulfonate, acetate, citrate, malonate, tartarate, succinate, benzoate, ascorbate, etoglutarate, and glycerophosphate. Suitable inorganic salts may also be formed, including hydrochloride, hydrobromide, sulfate, nitrate, bicarbonate, and carbonate salts.
Pharmaceutically acceptable salts may be obtained using standard procedures well known in the art, for example by reacting a sufficiently basic compound such as an amine with a suitable acid affording a physiologically acceptable anion. Alkali metal (for example, sodium, potassium or lithium) or alkaline earth metal (for example calcium) salts of carboxylic acids can also be made.
xe2x80x9cPharmaceutically acceptable saltsxe2x80x9d refers to those salts which possess the biological effectiveness and properties of the parent compound and which are not biologically or otherwise undesirable.
Compounds of the present invention can conveniently be administered in a pharmaceutical composition containing the compound in combination with a suitable excipient, the composition being useful in combating viral infections. Pharmaceutical compositions containing a compound appropriate for antiviral use are prepared by methods and contain excipients which are well known in the art. A generally recognized compendium of such methods and ingredients is Remington""s Pharmaceutical Sciences by E. W. Martin (Mark Publ. Co., 15th Ed., 1975).
The compounds and compositions of the present invention can be administered parenterally (for example, by intravenous, intraperitoneal or intramuscular injection), topically, orally, intravaginally, or rectally, depending on whether the preparation is used to treat internal or external viral infections.
For oral therapeutic administration, the active compound may be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 0.1% of active compound. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 2 to about 60% of the weight of a given unit dosage form. The amount of active compound in such therapeutically useful compositions is such that an effective dosage level will be obtained.
The tablets, troches, pills, capsules, and the like may also contain the following: binders such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, fructose, lactose or aspartame or a flavoring agent such as peppermint, oil of wintergreen, or cherry flavoring may be added. When the unit dosage form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier, such as a vegetable oil or a polyethylene glycol. Various other materials may be present as coatings or to otherwise modify the physical form of the solid unit dosage form. For instance, tablets, pills, or capsules may be coated with gelatin, wax, shellac or sugar and the like. A syrup or elixir may contain the active compound, sucrose or fructose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring such as cherry or orange flavor. Of course, any material used in preparing any unit dosage form should be pharmaceutically acceptable and substantially non-toxic in the amounts employed. In addition, the active compound may be incorporated into sustained-release preparations and devices.
The compounds or compositions can also be administered intravenously or intraperitoneally by infusion or injection. Solutions of the active compound or its salts can be prepared in water, optionally mixed with a nontoxic surfactant. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, triacetin, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
Pharmaceutical dosage forms suitable for injection or infusion can include sterile aqueous solutions or dispersions or sterile powders comprising the active ingredient which are adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes. In all cases, the ultimate dosage form should be sterile, fluid and stable under the conditions of manufacture and storage. The liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions or by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, buffers or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filter sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze drying techniques, which yield a powder of the active ingredient plus any additional desired ingredient present in the previously sterile-filtered solutions.
For topical administration, the present compounds may be applied in pure form, i.e., when they are liquids. However, it will generally be desirable to administer them to the skin as compositions or formulations, in combination with a dermatologically acceptable carrier, which may be a solid or a liquid.
Useful solid carriers include finely divided solids such as talc, clay, microcrystalline cellulose, silica, alumina and the like. Useful liquid carriers include water, alcohols or glycols or water-alcohol/glycol blends, in which the present compounds can be dissolved or dispersed at effective levels, optionally with the aid of non-toxic surfactants. Adjuvants such as fragrances and additional antimicrobial agents can be added to optimize the properties for a given use. The resultant liquid compositions can be applied from absorbent pads, used to impregnate bandages and other dressings, or sprayed onto the affected area using pump-type or aerosol sprayers. Thickeners such as synthetic polymers, fatty acids, fatty acid salts and esters, fatty alcohols, modified celluloses or modified mineral materials can also be employed with liquid carriers to form spreadable pastes, gels, ointments, soaps, and the like, for application directly to the skin of the user.
Examples of useful dermatological compositions which can be used to deliver the compounds of formula I to the skin are known to the art; for example, see Jacquet et al. (U.S. Pat. No. 4,608,392), Geria (U.S. Pat. No. 4,992,478), Smith et al. (U.S. Pat. No. 4,559,157) and Wortzman (U.S. Pat. No. 4,820,508).
Useful dosages of the compounds of formula I can be determined by comparing their in vitro activity, and in vivo activity in animal models. Methods for the extrapolation of effective dosages in mice, and other animals, to humans are known to the art; for example, see U.S. Pat. No. 4,938,949.
The compound is conveniently administered in unit dosage form; for example, containing 5 to 1000 mg, conveniently 10 to 750 mg, most conveniently, 50 to 500 mg of active ingredient per unit dosage form. The desired dose may conveniently be presented in a single dose or as divided doses administered at appropriate intervals, for example, as two, three, four or more sub-doses per day. The sub-dose itself may be further divided, e.g., into a number of discrete loosely spaced administrations; such as multiple inhalations from an insufflator or by application of a plurality of drops into the eye.
For internal infections, the compositions can be administered orally or parenterally at dose levels, calculated as the free base, of about 0.1 to 300 mg/kg, preferably 1.0 to 30 mg/kg of mammal body weight, and can be used in man in a unit dosage form, administered one to four times daily in the amount of 1 to 1000 mg per unit dose.
For parenteral administration or for administration as drops, as for eye infections, the compounds are presented in aqueous solution in a concentration of from about 0.1 to about 10%, more preferably about 0.1 to about 7%. The solution may contain other ingredients, such as emulsifiers, antioxidants or buffers.
Generally, the concentration of the compound(s) of formula I in a liquid composition, such as a lotion, will be from about 0.1-25 wt-%, preferably from about 0.5-10 wt-%. The concentration in a semi-solid or solid composition such as a gel or a powder will be about 0.1-5 wt-%, preferably about 0.5-2.5 wt-%.
The exact regimen for administration of the compounds and compositions disclosed herein will necessarily be dependent upon the needs of the individual subject being treated, the type of treatment and, of course, the judgment of the attending practitioner.
The antiviral activity of a compound of the invention can be determined using pharmacological models which are well known to the art, or using Test A described below.
The compounds of formula (I) and pharmaceutically acceptable salts thereof are useful as antiviral agents. Thus, they are useful to combat viral infections in animals, including man. The compounds are generally active against herpesviruses, and are particularly useful against the varicella zoster virus, the Epstein-Barr virus, the herpes simplex virus, the human herpesvirus type 8 (HHV-8) and the cytomegalovirus (CMV).
While many of the compounds of the present invention have shown activity against the CMV polymerase, these compounds may be active against the cytomegalovirus by this or other mechanisms of action. Thus, the description below of these compounds"" activity against the CMV polymerase is not meant to limit the present invention to a specific mechanism of action.
The HCMV polymerase assay is performed using a scintillation proximity assay (SPA) as described in several references, such as N. D. Cook, et al., Pharmaceutical Manufacturing International, pages 49-53 (1992); K. Takeuchi, Laboratory Practice, September issue (1992); U.S. Pat. No. 4,568,649 (1986); which are incorporated by reference herein. Reactions are performed in 96-well plates. The assay is conducted in 100 xcexcl volume with 5.4 mM HEPES (pH 7.5), 11.7 mM KCl, 4.5 mM MgCl2, 0.36 mg/ml BSA, and 90 nM 3H-dTTP. Assays are run with and without CHAPS, (3-[(3-Cholamidopropyl)-dimethylammonio]-1-propane-sulfonate) at a final concentration of 2 mM. HCMV polymerase is diluted in enzyme dilution buffer containing 50% glycerol, 250 mM NaCl, 10 mM HEPES (pH 7.5), 100 xcexcg/ml BSA, and 0.01% sodium azide. The HCMV polymerase, which is expressed in recombinant baculovirus-infected SF-9 cells and purified according to literature procedures, is added at 10% (or 10 xcexcl) of the final reaction volume, i.e., 100 xcexcl. Compounds are diluted in 50% DMSO and 10 xcexcl are added to each well. Control wells contain an equivalent concentration of DMSO. Unless noted otherwise, reactions are initiated via the addition of 6 nM biotinylated poly(dA)-oligo(dT) template/primer to reaction mixtures containing the enzyme, substrate, and compounds of interest. Plates are incubated in a 25xc2x0 C. or 37xc2x0 C. H2O bath and terminated via the addition of 40 xcexcl/reaction of 0.5 M EDTA (pH 8) per well. Reactions are terminated within the time-frame during which substrate incorporation is linear and varied depending upon the enzyme and conditions used, i.e., 30 min. for HCMV polymerase. Ten xcexcl of streptavidin-SPA beads (20 mg/ml in PBS/10% glycerol) are added following termination of the reaction. Plates are incubated 10 min. at 37xc2x0 C., then equilibrated to room temperature, and counted on a Packard Topcount. Linear regressions are performed and IC50""s are calculated using computer software.
A modified version of the above HCMV polymerase assay is performed as described above, but with the following changes: Compounds are diluted in 100% DMSO until final dilution into assay buffer. In the previous assay, compounds are diluted in 50% DMSO. 4.5 mM dithiotherotol (DTT) is added to the polymerase buffer. Also, a different lot of CMV polymerase is used, which appears to be more active resulting in a more rapid polymerase reaction. Results of the testing of representative compounds of formula I in this assay are shown in Table 1 below.
At the table, the term xe2x80x9cn.d.xe2x80x9d refers to xe2x80x9cnot determinedxe2x80x9d.
The compounds and their preparation of the present invention will be better understood in connection with the following examples, which are intended as an illustration of and not a limitation upon the scope of the invention.